In the addition of reagents in small quantities to micro reaction systems, it is important that all the reagent be delivered and that none be left on the sides of the reaction vessel. This is especially true when the added reagent is part of an eventual detection system for studying the reaction. For example, in the addition of radioactive labelled materials to a reaction vessel, where the final measurement is the amount of radioactivity remaining in the vessel, perhaps in the form of a precipitate or complex bound to the surface of the vessel, it is highly desirable that no labelled material be splashed or otherwise be deposited onto the sides of the vessel, which would lead to a higher than expected count. This is especially true if the vessel is placed directly into the counting apparatus as in the case of a tube.
This problem has direct relevance to radioimmune assays conducted in small tubes, where volumes are small and accurate counts are required. The basic premise behind any radioimmune assay is that an antigen or antibody, is allowed to combine with its respective radioactively-labelled antibody or antigen, as the case may be, and that the resultant labelled complex is isolated, the radioactivity measured and, from this figure and comparison with measurements taken on standard materials, the amount of original material calculated.
One particular type of assay that employs an extension of this concept is the so-called "sandwich" assay employing protein-coated tubes. In this assay, an antibody to the desired antigen is first absorbed from solution onto the inner surface of a glass or plastic tube. The remainder of the solution is removed and the tube washed. Both glass and plastic will readily absorb proteins onto their surfaces and after the washing a "coat" of antibody is left on the tube. The samples to be tested are then placed in the tubes and any antigen present in the sample will be complexed by the antibody on the tube. After removal of the sample and washing, the tubes are treated with radioactively-labelled antibody and it will be readily seen that where antigen is present, labelled antibody will be complex in a "sandwich" onto the tube. After washing, the tube is placed directly in a counter and the number of counts in excess of a known negative standard will give an estimate of the quantity of antigen present in the original sample.
Because of the aforementioned adsorptive properties of the tube material, any radioactive protein that is splashed or otherwise placed on the sides of the tube rather than contacting the already-coated portion of the tube may absorb to the inside of the tube or dry onto the wall of the tube. This labelled protein will not readily be washed off and will tend to leave labelled protein on the tube where it has not reacted with the antigen, resulting in a higher count than expected. Such higher counts will be especially detrimental in samples in which there is no antigen and which might normally be expected to give low counts. When this occurs, the result is a false positive reaction.
Whilst it is relatively easy to ensure that no splashing occurs on the addition of reagents, the actual reagent addition requires dexterity and is time-consuming and tedious. Because radioimmune assays are becoming very useful for diagnostic purposes and many hundreds of tests have to be performed in a day, it is desirable that false positives be reduced to a minimum and yet the maximum number of tests be performed.